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AcceptedPrepri n
tBiofuels and Environmental Biotechnology Biotechnology and Bioengineering
DOI 10.1002/bit.23160
Culture of Microalgae Chlorella minutissima for Biodiesel Feedstock
Production
Haiying Tang, Meng Chen, M. E. D. Garcia, Nadia Abunasser, K. Y. Simon Ng, and
Steven O. Salley
Department of Chemical Engineering and Materials Science,
Wayne State University,
5050 Anthony Wayne Drive, Detroit, MI 48202, USA
Corresponding author: Steven O. Salley
Department of Chemical Engineering and Materials Science
Wayne State University
5050 Anthony Wayne Drive
Detroit, Michigan 48202
Tel: (313) 577-5216
Fax: (313) 577-3810
Email: [email protected]
© 2011 Wiley Periodicals, Inc.
Received November 1, 2010; Revised February 25, 2011; Accepted March 25, 2011
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tAbstract
Microalgae are among the most promising of non-food based biomass fuel feedstock
alternatives. Algal biofuels production is challenged by limited oil content, growth rate, and
economical cultivation. To develop the optimum cultivation conditions for increasing biofuels
feedstock production, the effect of light source, light intensity, photoperiod, and nitrogen
starvation on the growth rate, cell density, and lipid content of Chlorella minutissima were studied.
The fatty acid content and composition of Chlorella minutissima were also investigated under the
above conditions. Fluorescent lights were more effective than red or white light-emitting diodes
(LEDs) for algal growth. Increasing light intensity resulted in more rapid algal growth, while
increasing the period of light also significantly increased biomass productivity. Our results
showed that the lipid and triacylglycerol (TAG) content were increased under N starvation
conditions. Thus, a two-phase strategy with an initial nutrient-sufficient reactor followed by a
nutrient deprivation strategy could likely balance the desire for rapid and high biomass generation
(124 mg/L) with a high oil content (50%) of Chlorella minutissima to maximize the total amount
of oil produced for biodiesel production. Moreover, methyl palmitate (C16:0), methyl oleate
(C18:1), methyl linoleate (C18:2), and methyl linolenate (C18:3) are the major components of
Chlorella minutissima derived FAME, and choice of light source, intensity, and N starvation
impacted the FAME composition of Chlorella minutissima. The optimized cultivation conditions
resulted in higher growth rate, cell density, and oil content, making Chlorella minutissima a
potentially suitable organism for biodiesel feedstock production.
Keywords: microalgae, Chlorella minutissima, algae cultivation, biodiesel production
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t1 Introduction
Developing biomass feedstocks for biofuel production is vital for our energy security and
national economy, as well as for our environment. Biodiesel is a renewable fuel defined as alkyl
monoesters of long chain fatty acids, which has been widely used in diesel engines especially in
Europe and the USA (Ma and Hanna 1999; Vasudevan and Briggs 2008). However, the cost and
availability of traditional feedstocks for biodiesel has become a critical concern. Algae are among
the most promising non-food-crop-based biomass feedstocks (Chisti 2007). Some green algae
species have been reported to have a high lipid content and potentially may serve as a viable
biodiesel oil source , such as Nannochloropsis sp. (Gouveia and Oliveira 2009; Rodolfi et al.
2009), Tetraselmis sueccica (Rodolfi et al. 2009), Chlorella sp. (Rodolfi et al. 2009), and
Scenedesmus sp. (Matsunaga et al. 2009; Rodolfi et al. 2009). However, at present there are a very
limited number of large-scale commercial operations that grow and harvest algae and these
mainly produce relatively high value products such as food supplements and none economically
produce algae with the potential for biofuel use. Several factors limit algal production of oil and
hydrocarbons for biofuels: existing algae strains do not possess both high oil content and a high
growth rate and they cannot be grown to high cell densities; the design of large scale production
systems (open ponds and photobioreactor systems) have not been fully optimized; and harvesting
of algae and extraction and processing of oil remain a challenge. Moreover, the key properties of
biodiesel are highly dependent on the fatty acid (FA) composition of the original triacylglycerol
(TAG) (Tang et al. 2008). A high oil-producing species with an optimized FA profile for
production of high quality biodiesel is desirable for a microalgal biodiesel which meets standards
for user acceptance.
Chlorella minutissima is an eukaryotic alga, with relative fast growth and easy cultivation,
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tand the levels of amino acids and polyunsaturated fatty acids in Chlorella minutissima are high,
which could be potentially useful in health foods and pharmaceuticals (Seto et al. 1984). There
have been few studies focused on biofuel feedstock production using Chlorella minutissima
(Bhatnagar et al. 2010). Moreover, Chlorella minutissima is an extremely high-CO2-tolerent alga,
which may be developed to use waste CO2 for algal cultivation (Papazi et al. 2008). Therefore,
one freshwater strain, Chlorella minutissima, was chosen in the present research to find the best
conditions of cultivation, including light source, light intensity, photoperiod, and nutrient, for high
growth rate and cell density, oil content, and suitable FA profile for biodiesel production.
2. Materials and methods
2.1 Algal cultures
Chlorella minutissima (UTEX 2219) was obtained from the University of Texas at Austin
(UTEX). It is a green unicellular alga and has a cell size of about 2 ~ 5 µm. The cells were
cultured in Modified Bold 3N medium, consisting of 850 mL of DI H2O, 6 mL of P-IV metal
solution (0.75 g/L of Na2EDTA·2H2O (Sigma ED255), 0.097 g/L of FeCl3·6H2O (Sigma 1513),
0.041 g/L of MnCl2·4H2O (Baker 2540), 0.005 g/L of ZnCl2 (Sigma Z-0152), 0.002 g/L of
CoCl2·6H2O (Sigma C-3169), and 0.004 g/L of Na2MoO4·2H2O (J.T. Baker 3764)), 30 mL of 10
g/400 mL of NaNO3 (Fisher BP360-500), 10 mL of 1 g/400 mL of CaCl2·2H2O (Sigma
C-3881), 10 mL of 3 g/400 mL of MgSO4·7H2O (Sigma 230391), 10 mL of 3 g/ 400 mL of
K2HPO4 (Sigma P 3786), 10 mL of 7 g/400 mL of KH2PO4 (Sigma P 0662), 10 mL of 1
g/400mL of NaCl (Fisher S271-500), 40 mL of soil-water (1 teaspoon of garden soil (SCHULTZ
enriched garden soil, St. Louis, Missouri) was added in 200 mL of DI water), 1 mL of vitamin
B12 (0.1 mM of Vitamin B12 (cyanocobalamin, (Sigma V-6629) was added to 200 mL of 50 mM
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tHEPES buffer (Sigma H-3375) and the pH was adjusted to 7.8), 1 mL of biotin vitamin solution
(0.1 mM of biotin (Sigma V-4639) was added in 200 mL of 50mM HEPES buffer (Sigma
H-3375) and the pH was adjusted to 7.8), and 1 mL of thiamine vitamin solution (6.5 mM of
thiamine (Sigma T-1270) was added in 50 mL of 50mM HEPES buffer (Sigma H-3375) and the
pH was adjusted to 7.8). The total pH of Modified Bold 3N medium is 6.2. The components of
N-depleted medium were the same as the above Modified Bold 3N medium except excluding
NaNO3 and soil-water. In other experiments, it was found that elimination of vitamins from
Modified Bold 3N medium did not affect growth of Chlorella minutissima (data not shown).
Algae cultures were set up in 650 mL culture flasks (4.45×10.16×14.38 cm3, Greiner
Bio-One Gmbh, Germany) with a feed gas flow rate of 60 mL/min bubbled air/CO2 mixture
through an aquafizz 1 inch air stone (Petsolutions, Beavercreek, OH). The culture flask was
illuminated with external lights that were automatically turned on/off to simulate a circadian
cycle. Images of culture flask have been showed elsewhere (Tang et al. 2010). The temperature
of the reactor was maintained at 23 oC. The pH was not controlled. Three different light sources,
including a total of 20 strips of white light-emitting diodes (LEDs are supplied in strips, with 24
LEDs per 9 inch strip (0.8 watts), Super Bright LEDs, Inc. St. Louis, Missouri), 20 strips of red
LEDs, and one fluorescent light (one 12 inch 8 watts, Phillips), with the same initial light
intensity (100 µE/ (m2
s)) were evaluated for their effect on algal growth rate. Moreover, the
initial light intensities of 100, 200, 350 and 400 µE/(m2
s) were attained using one, two, three,
and four of fluorescent lights, respectively. Photoperiods of 15 hr light: 9 hr dark, 12 hr light: 12
hr dark, and 24 hr light: 0 hr dark was investigated with the initial light intensity of 200 µE/ (m2
s).
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t2.2 Growth analysis
Algal growth was measured by daily changes in both optical density at 680 nm with a
spectrophotometer (Evolution 60, Thermo Scientific, Waltham, MA) and cell numbers counted
under an OLYMPUS BX51 microscope (Optical Analysis Corp., Nashua, NH) with a
haemacytometer (Hausser Scientific, Horsham, PA). One unit of OD680 corresponded to 4 × 107
cells/mL. Determining the dry weight of Chlorella minutissima is by drying the algae for 2 hr in
an oven at 110°C. The temperature and pH were recorded every day using an Oakton pH 110
advanced portable pH meter (Euthech Instrument, Singapore).
The light intensity was measured at eighteen fixed spots on the side of the flask opposite
the light source using a light meter (LI-1400, LI-COR Inc., USA), and the average of eighteen
readings of light intensity was obtained at the same time daily.
The inorganic nitrogen content was quantified in the forms of nitrate using a portable
spectrophotometer (HACH DR 2800, Loveland, Colorado). Briefly, 5 mL algae culture was
collected, and then was centrifuged and the supernatant was transferred to another clean tube. A
one mL sample was analyzed for nitrogen content using reaction kit Nitrate (0-30 mg/L NO3-N,
Hach Lange GmbH).
2.3 Lipid analysis
2.3.1 Lipid extraction
The lipid extraction method was based on the methods of Bligh & Dyer (Bligh and Dyer
1959). The algae suspension was centrifuged at 5000 rpm (Eppendorf centrifuge 5804R, Germany)
for 10 min; the upper layer of medium was removed and the concentrated algae was obtained, and
subsequently frozen at -20 °C for 24 hr. After freezing, the about 4 g of algae was disrupted with
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tglass beads (Sigma, St. Louis) in a vortex mixer (Scientific Industries Inc, Bohemia, NY) for 10
min. The lipid was then extracted with 4 mL of chloroform: methanol (2:1), and then the extracted
lipid was centrifuged at 5000 rpm (Eppendorf centrifuge 5804R, Germany) for 10 min to separate
into three layers. The upper layer (methanol layer including water) was removed and the
chloroform layer including lipid was collected. The residues were subjected to repeated extraction
three times. All extracts obtained were then mixed together to form the crude oil extract from
which the chloroform was evaporated to yield the resultant algae oil, and finally weighed to give
the total extracted organic content (TEO). The TEO content was evaluated by its weight relative
to the weight of dry algal biomass. The definition of TEO content is the same as the total lipid
content quantified gravimetrically by others (Bhatnagar et al. 2010; Gouveia and Oliveira 2009;
Liu et al. 2008).
2.3.2 FAME analysis
The FAME was prepared from the resultant algae oil. The sodium methylate catalyst
(CH3ONa, 0.1 M/MEOH, 1.5 mL), and tetrahydrofuran solubility improver (5 mL, EMD
chemical Inc. Gibbstown, NJ) were added to the algal oil and heated at 110 °C for 5 hours in a
reactor (General purpose vessel 4744, Parr Instrument Company, Moline, IL). After the reaction
was completed, the products were cooled to room temperature, and solvent was evaporated. The
content and composition of FAME was further analyzed via GC-MS.
The FAME sample was dissolved in 1 mL of heptane, then a 100 L sample was mixed
with 100 L of ethyl arachidate (~ 10 mg/mL, Nu-Chek Prep, Inc., Elysian, Minnesota), which
was used as an internal standard. Finally, 900 L of heptane was added to the vial. The prepared
sample was analyzed using a PerkinElmer Clarus 500 GC-mass spectrometer (GC-MS) with a
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tsplit automatic injector and a Rtx-WAX (Restek, Bellefonte, PA) column (length: 60 meters; ID:
0.25 mm, coating: 0.25 µm). Details of the procedure have been described elsewhere (Tang et al.
2008). The column was held at 120 ºC for 1 minute and ramped to 240 ºC at 20 ºC/min, and it was
then held at 240 ºC for 13 minutes. The transfer line between GC and MS was kept at 240 ºC. The
FAME contents were determined by comparing their peak areas with that of the internal standard
(EEC20:0). The total FAME content was evaluated by its weight relative to the weight of dry algal
biomass.
2.3.3 FFA, TAG, DG, and MG analysis
A high performance liquid chromatographic (HPLC) method was used to determine the
free fatty acid (FFA), TAG, diglyceride (DG), and monoglyceride (MG) content in the resultant
algae oil (Foglia and Jones 1997). The HPLC analysis was conducted using a PerkinElmer Series
200 with an Altech 3300 Evaporative Light Scattering Detector (ELSD) and a Perkin Elmer
Brownlee Validated Cyano column (250×4.6mm, 5µm) with guard column (7.5×4.6mm, 5µm) as
the stationary phase and a flow rate of 1.0 mL/min. Mobile phase solvents were hexane with 0.4%
acetic acid (Phase A) and methyl t-butyl ether with 0.4% acetic acid (Phase B). A detailed elution
scheme is given in Table 1. The column temperature was set to 25 °C and the injection volume was
20 µL.
3. Results
3.1 Effect of light sources
Chlorella minutissima flask cultivations were performed with 100 µE/(m2
s) initial light
intensity of red LEDs, white LEDs, and fluorescent lights under a photoperiod of 24 hr light, 4%
CO2, temperature of 23 ºC, and pH of between 6.4 and 7.0. The fastest growth of Chlorella
minutissima was observed with florescent light with the cell concentration reaching 2.3×108
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tcells/mL after 13-day cultivation, followed by white LEDs (1.7×10
8 cells/mL) and red LEDs
(1.7×108 cells/mL). Moreover, the corresponding light intensity with red LEDs, white LEDs, and
fluorescent lights was found to be decreased with the increasing cell density as a function of time
(Fig. 1). The varying light intensity corresponding to cell density is consistent with three types of
light sources. The higher biomass content (dry weight) with fluorescent light was greater than
with the white and red LEDs (Table 2).
There was no significant difference in total FAME (TFAME) content (~ 10%) with three
types of light sources (Table 2). Chlorella minutissima methyl esters were predominantly methyl
linolenate (C18:3), methyl palmitate (C16:0), methyl linoleate (C18:2), and methyl oleate (C18:1),
with minor fractions of methyl palmitoleate (C16:1), methyl hexadecadienoate (C16:2), and
methyl hexadecatrienoate (C16:3). The total unsaturated FAME content didn’t change with
different light source, and it constituted ~ 82% of the known total FAME fraction (Table 2).
However, for methyl linolenate (C18:3) and methyl linoleate (C18:2), there were significant
differences with florescence light, white LEDs, and red LEDs as light source: methyl linolenate
(C18:3) composition with red LEDs was significantly decreased from ~ 30% with florescence
light and white LEDs to ~ 20%; while methyl linoleate (C18:2) composition was increased.
3.2 Effect of light intensity
Fluorescent light with 100, 200, 350, and 400 µE/(m2s) initial light intensities were utilized
for Chlorella minutissima cultivation with a photoperiod of 24 hr light, 4% CO2, and temperature
of 23 ºC. The results of the cell density and nitrate concentration as a function of time are shown
in Fig. 2. A significantly higher cell grow rate was observed with increasing light intensity of 100,
200 and 350 µE/ (m2
s), while no apparent difference in cell grow rate was found with light
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tintensity of between 350 and 400 µE/ (m
2s). The nitrate concentration declined continuously with
cultivation time, and was zero when the cell density reached a maximum value, indicating that
growth may have been nitrate limited. The biomass content of Chlorella minutissima increased
with increasing light intensity (Table 3) consistent with the growth shown in Figure 2.
The total FAME content by weight relative to the weight of dry algal biomass was not
significantly different for any light intensity. However, the fraction of saturated FAME with 200,
350, and 400 µE/ (m2s) of initial light intensity was higher than 100 µE/ (m
2s) (~31% vs. ~22%),
which was related to the increasing methyl palmitate (C16:0) at the higher light intensity.
3.3 Effect of photoperiod
Figure 3 shows the growth of Chlorella minutissima as a function of illumination time with
three different photoperiods of 12 hr light: 12 hr dark, 15 hr light: 9hr dark, and 24 hr light: 0 hr
dark. Each algal culture was illuminated by fluorescent light with a light intensity of 200 µE/(m2
s), 4% CO2, temperature of 23 ºC, and pH of between 6.4 and 7.0 in culture flasks. For a given
total time of illumination, the photoperiods of 12 hr light: 12 hr dark and 15 hr light: 9hr dark
achieved a higher cell density as compared to the photoperiod of 24 hr light: 0 hr dark. However,
the total biomass productivity with continuous light was higher than with the other photoperiods
(Table 4). Table 4 also shows that the photoperiod had no significant influence on TFAME content
(~10%) and composition of Chlorella minutissima (~77% unsaturated FAME).
3.4 Effect of N starvation
Starting from a culture grown in nutrient-sufficient medium which achieved an
exponential growth phase after 4-day cultivation (1.2×108 cells/mL), and N-deprivation
cultivation was achieved by centrifuging the culture and replacing the medium with the same
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tvolume of N-depleted medium. As shown in Fig. 4, the cells of Chlorella minutissima still
exhibited exponential growth under N-depleted medium and obtained a cell density of 2.5 ×108
cells/mL after 5 additional days of cultivation. The biomass content increased from 560 mg/L
(before N-deprivation cultivation) to 1240 mg/L (after N-deprivation cultivation) (Table 5).
The TEO content determined by gravimetric method was 35.3% before N-depleted
medium cultivation (1.2×108 cells/mL of cell concentration at the exponential growth phase),
while the TEO content was up to 50% after N-deprived medium cultivation (Table 5).
N-deprivation also led to a significant increase of the TFAME content from ~ 6% to 18%.
Moreover, methyl linolenate (C18:3) composition was significant decreased from ~27% to 17%,
while methyl oleate (C18:1) composition was increased from 21% to 32%; however, the total
unsaturated composition had no significant change under N-deprivation.
Table 6 shows FFA, TAG, DG, and MG content (% dry weight biomass) determined by
HPLC analysis. The total oil content including FFA, TAG, DG, and MG content was significantly
increased from around 12% to 20% in N-deprived medium. TAG and MG content were found to
be increased from ~ 1% to 9% and ~ 2% to 8%, respectively; while the FFA content was decreased
from 9% to 2% in the N-deprived medium. The TFAME content ( ~ 18%) determined by GC-MS
and total FFA, TAG, DG and MG content (~ 20%) determined by HPLC are much less than the
TEO content (50%) determined by gravimetric Bligh & Dyer method. This is likely because the
total extracted lipids include fatty acids, triglycerides, chlorophyll, and the other components.
4 Discussion
Algal production systems included open pond/raceway systems (outdoor cultivation), and
photobioreactor (PBR) systems (indoor cultivation). Open ponds are the less expensive than PBR
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tsystem, but the disadvantage of open ponds system is ease of contamination, low productivity,
high harvesting cost, and large volume of water loss (Hu et al. 2006; Shen et al. 2009; Xu et al.
2009). A PBR is a closed system that reduces the chances of contamination, allows precise
environmental control, operates as a continuous process for harvesting and producing of algae
biomass to improve efficiency; however, PBRs have a higher capital cost than open pond systems
as well as a likely high energy cost associated with cooling (Pulz 2001). The main factor affecting
microalgae cell growth in photoautotrophic cultivation is light. Algal growth is limited by
insufficient light, but also may be inhibited by over exposure to light. Our results show that red
LEDs, white LEDs, and fluorescent lights are efficient for Chlorella minutissima photosynthesis
but with a higher growth rate with fluorescent light than with white and red LEDs and comparable
intensity. This observation is contrary to a previous study where fluorescent lights, red LEDs,
and white LEDs had similar growth rates with Dunaliella tertiolecta (Tang et al. 2010). Moreover,
our findings indicated that a higher light intensity (350 µE/(m2
s) was more efficient to improve
algae Chlorella minutissima biomass density than lower light intensity (100 ~ 200 µE/(m2
s)), and
the biomass density could reach up to 1000 mg/L dry mass after 7 days of cultivation, but a light
intensity between 350 and 400 µE/(m2
s) could not further significantly increase growth rate. An
earlier study showed that the density of Chlorella minutissima was 386 mg/L dry mass with ~23
µE/(m2s) light intensity and 5% CO2 concentration after 4 days of cultivation, while the biomass
density was 66 mg/L dry mass with ~ 3 µE/(m2s) light intensity (Seto et al. 1984). Bhatnagar et al.
(Bhatnagar et al. 2010) found that the biomass density was 73 mg/L dry mass under ambient CO2
concentration in standard BG 11 medium under phototrophic conditions (30 µE/(m2
s) light
intensity in 6/18 light/dark cycle) after 10 days of growth, while 379 mg/L dry mass under
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tmixotrophic condition (30 µE/(m
2s) light intensity and BG 11 medium supplemented with
glucose), and 140 mg/L under heterotrophic condition (BG 11 medium supplemented with
glucose in dark). In our conditions, the best biomass productivity of Chlorella minutissima was
~143 mg/L/day under continuous illumination with 350 µE/(m2s) fluorescent light combined with
4% CO2, and temperature of 23 ºC, and the biomass productivity was significantly increased as
compared with Vazhappilly and Chen’s results (~89.4 mg/L/day) (Vazhappilly and Chen 1998).
The dry biomass productivity (143 mg/L/day) is higher compared to 90 mg/L/day (dry biomass)
reported for both Neochloris oleoabundans and Nannochloropsis sp. in an outdoor Raceways
Pond (Gouveia and Oliveira 2009), a maximum biomass productivity of 47 mg/L/day with 11%
(w/w) of lipid content (da Silva et al. 2009) for Neochloris oleoabundans in an open race pond
(375 L) (da Silva et al. 2009), which were used as a suitable feedstocks for biodiesel production.
Moreover, Jiang et al. compared the cultivation of Dunaliella salina between outdoors and in
PBR and found that cultivation of algae in the PBR ( 24 mg/L/day of biomass productivity) was
more effective than that outdoors (20 mg/L/day) at the end of the logarithmic phase (Jiang and
Zhu 2010). Since our results are for a PBR system, they may not be directly transferable to
outdoor cultivation conditions.
Light penetration is dependent on the geometry of the reactor and the cell density of the
culture. Our results showed the effective light intensity decreased to zero at the vessel wall
opposite the light source as the cell concentration increased (Fig. 1). Light limitation was one of
the factors which could decrease algal photosynthetic rate in the log phase. While light
determined the growth rate, nitrate consumption appears to limit overall yield (Fig. 2). The
exponential growth period (log phase) was shorter under higher light intensity, i.e., at 350 and 400
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tµE/(m
2s), as compare to lower light intensity such as (100 and 200 µE/(m
2s) ). However, it is
worth noting that algae cells in N-depleted medium still had a higher growth rate when the
nitrogen starvation-stage culture process began from algae cells which were in the beginning of
log growth phase (Fig. 4). It is possible that nitrogen was accumulated within the algal cells
during the exponential phase period, and then was consumed during the algae growth in the
N-depleted medium (Lourenco et al. 1998).
The major factors affecting lipid accumulation are nutrient starvation, salinity,
growth-medium pH, temperature, and light intensity, followed by growth phase and/or aging of
the culture (Hu et al. 2008). Our results indicated that nitrogen starvation was an effective method
to improve TEO (total lipid) content and TAG content in the algae Chlorella minutissima. The
light source, light intensity, and photoperiod had no significant effect on TEO content and FAME
content. These results are in agreement with other studies, which reported that the lipid content of
Nannochloropsis sp., Neochloris oleoabundans, Scenedesmus obliqus, and Chlorella vulgaris
was increased from 29 to 60 %, 16 to 34 %, 22 to 55 %, and 25 to 50 % under N starvation,
respectively (Gouveia et al. 2009; Li et al. 2008; Rodolfi et al. 2009; Widjaja et al. 2009). Rodolfi
et al. (Rodolfi et al. 2009) also reported that nitrogen starved cells can accumulate up to 85% lipid
in its biomass while the typical content of cultures in the exponential phase was only about 5%.
However, the increased oil content of the algae may not lead to increased overall productivity of
oil because higher levels of oil in the cells are often more than offset by lower rates of cell growth
(Sheehan et al. 1998). Nitrogen level in the medium has an inverse influence on microalgal
growth and lipid storage (Li et al. 2008; Widjaja et al. 2009). A two-stage strategy with an initial
nutrient-sufficient reactor followed by a nutrient deprivation reactor can make biomass density of
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tChlorella minutissima to increase up to 1240 mg/L with 50% of total lipid content under
N-depleted condition, which was achieved at 400 µE/(m2
s) light intensity of continuous
illumination and 4% CO2 after 9- day cultivation in our study. Bhatnagar et al. (Bhatnagar et al.
2010) reported that the biomass of Chlorella minutissima was 73 mg/L, 379 mg/L, and 140 mg/L
under phototrophic, mixotrophic, and heterotrophic conditions, respectively, while the
corresponding lipid contents was 3.9%, 56.4%, and 16.5%. Therefore, the two-phase strategy with
an initial nutrient-sufficient reactor followed by a nutrient deprivation strategy may balance the
need for rapid and high biomass generation with high oil content.
GC-MS analysis demonstrates that FA profile from Chlorella minutissima mainly contains
the long chain and un-branched fatty acids with C16 to C18 carbons, thus, Chlorella minutissima
could be considered as a potential organism for biodiesel production. Among the unsaturated fatty
acids of Chlorella minutissima, special attention should be taken in relation to the about 30% of
linolenic methyl ester (18:3), which may result in the poor stability of fuel. Moreover, an
important factor affecting the FA profile of algae is light intensity. Earlier study showed that a
higher light intensity can increase TAG accumulation and saturated FA, while mainly polar lipids
(phospholipids and glycolipids), structurally and functionally associated with cell membranes, are
formed under lower light intensity (Hu et al. 2008). This is consistent with our results that the
lower light intensity (100 µE/ (m2s)) led to increases in the unsaturated FA as compared with the
higher light intensity. Seto et al. (Seto et al. 1984) also reported that light intensity affected the
composition of FA of Chlorella minutissima. However, the FA composition didn’t significantly
change with 200 to 400 µE/ (m2
s) of light intensity in our study. Interestingly, our results
indicated the light source and N-starvation also influenced the FA profile of Chlorella minutissima:
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tlinolenic acid (C18:3) composition of Chlorella minutissima was significantly decreased with red
LEDs as light source and under N-starvation conditions; but the total unsaturated FA didn’t
significantly change. It should be noted that the profile of FFA, TAG, DG, and MG was
significantly changed in N- starvation condition: The N-deprivation increases the TAG
accumulation, while FFA is formed in the nutrient-sufficient medium.
5 Conclusions
This study has demonstrated that maximum biomass productivity of Chlorella
minutissima can reach to ~143 mg/L/day under fluorescent light with 350 µE/ (m2
s) light
intensity combined with continuous light, 4% CO2, and temperature of 23 ºC. Chlorella
minutissima under N-deficient culture medium showed a great increase in lipid content and TAG
content, and Chlorella minutissima presents adequate desirable fatty acid profile. Therefore,
Chlorella minutissima may be suitable as raw material for biodiesel production
Acknowledgements
Financial support from National Institute of Food and Agriculture for this research is
gratefully acknowledged.
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AcceptedPrepri n
tList of Figure Captions:
Figure 1 Cell densities of Chlorella minutissima with red light-emitting diodes (LEDs), white
LEDs, and fluorescent lights as light source and corresponding light intensity as a
function of illumination time. The average of duplicate cultures is shown. Cultures
were illuminated under a photoperiod of 24 hr light with 100 µE/(m2s) initial light
intensity, 4% CO2, and temperature of 23 ºC.
Figure 2 Cell density of Chlorella minutissima with light intensity of 100, 200, 350, and 400
µE/(m2
s) and corresponding nitrate concentration in medium as a function of
illumination time. Cultures were illuminated by fluorescent light with a photoperiod
of 24 hr light, 4% CO2, and temperature of 23 ºC.
Figure 3 Cell density of Chlorella minutissima with different photoperiod as a function of
illumination time. Cultures were illuminated by fluorescent light with a light
intensity of 200 µE/(m2s), 4% CO2, and temperature of 23 ºC.
Figure 4 Growth curve of Chlorella minutissima with/without nitrogen starvation as a function
of illumination time. Cultures were illuminated by a photoperiod of 24 hr fluorescent
light with a light intensity of 400 µE/(m2s), 4% CO2, and temperature of 23 ºC.
AcceptedPrepri n
tList of Table Captions:
Table 1 Gradient condition of the HPLC method.
Table 2 Summary of fatty acid (FA) composition, biomass content, total extracted organic
content (TEO), and total fatty acid methyl ester (FAME) content of Chlorella
minutissima cultured with florescence light, white LEDs, and red LEDs after 14 days of
cultivation.
Table 3 Summary of FA composition, biomass content, TEO content, and total FAME content of
Chlorella minutissima with four levels of light intensity: 100, 200, 350, and 400 µE/ (m2
s) after 7 days of cultivation.
Table 4 Summary of FA composition, biomass content and productivity, TEO content, and total
FAME content of Chlorella minutissima with different photoperiod of 9 hr light: 15 hr
dark, 12 hr light: 12 hr dark, and 24 hr light: 0 hr dark, after 14 days of cultivation.
Table 5 Summary of FA composition, biomass content, TEO content, and total FAME content of
Chlorella minutissima with before and after nitrogen starvation cultivation.
Table 6 FFA, TAGs, DG, and MG content (% dry weight biomass) determined by HPLC
analysis.
AcceptedPrepri n
tTable 1
Step Time (min) A (%) B (%)
Equilibrium 15 100 0
1 5 100 0
2 15 20 80
3 17 20 80
17.1 17.1 100 0
5 27 100 10
AcceptedPrepri n
tT
able
2
Fat
ty a
cid
co
mp
osi
tio
n a (
%)
Flu
ore
scen
tW
hit
e L
ED
R
ed L
ED
C16:0
18.4
± 0
.4
18.2
3 ±
0.2
1
18.6
3 ±
0.9
3
C16:1
2.8
7 ±
0.2
5
1.4
3 ±
0.0
6
1.2
± 0
.26
C16:2
1.9
3 ±
0.2
1
0.7
7 ±
0.0
6
1.5
3 ±
0.2
1
C16:3
7.8
± 0
.26
10.9
± 0
.17
10.9
± 0
.17
C18:0
00
0
C18:1
18.9
7 ±
0.7
4
20.1
3 ±
0.1
5
21.9
33 ±
0.3
8
C18:2
19.6
3 ±
0.2
5
19.3
3 ±
0.1
5
27.3
7 ±
0.4
2
C18:3
30.3
7 ±
0.5
5
29.2
± 0
.1
19.5
3 ±
0.4
9
SF
A18.4
3 ±
0.4
18.2
3 ±
0.2
1
18.6
3 ±
0.9
3
UF
A81.5
7 ±
0.4
81.7
7 ±
0.2
1
81.3
7 ±
0.9
3
Bio
mas
s co
nte
nt
(dry
wei
gh
t, g
/L)
0.9
30
.85
0.8
7
Ex
trac
ted
org
anic
co
nte
nt b
(%
) 3
8.9
36
.73
4.4
TF
AM
E c
on
ten
t c(%
)9
.41
± 0
.47
1
1.3
5 ±
1.9
9
.99
± 0
.61
a P
erce
nta
ge
calc
ula
ted
bas
ed o
n t
he
tota
l k
no
wn
fat
ty a
cid
s.
b E
xtr
acte
d o
rgan
ic/
dry
cel
l w
eig
h ×
10
0%
c T
ota
l fa
tty
aci
d m
eth
yl
este
r (F
AM
E)/
dry
cel
l w
eig
ht
×1
00
%
AcceptedPrepri n
tT
able
3
Fat
ty a
cid
co
mp
osi
tio
n (
%)
10
0µ
E/m
2s
20
0µ
E/m
2s
35
0µ
E/m
2s
40
0µ
E/m
2s
C16:0
21.8
± 0
.75
30.1
± 1
.42
30.8
± 1
.78
29.7
± 1
.25
C16:1
2.9
± 0
.12
1.5
± 0
.21
1.3
± 0
.32
1.3
± 0
.35
C16:2
1.2
± 0
.15
0.3
± 0
.12
0.3
± 0
.15
0.4
± 0
.21
C16:3
8.3
± 0
.1
6.6
± 0
.51
6.1
± 0
.38
6.4
± 0
.51
C18:0
0.3
± 0
.06
1.2
± 0
.1
1.3
± 0
.1
1.3
C18:1
14.8
± 0
.46
13.5
± 1
.01
16 ±
0.6
1
17.5
± 0
.7
C18:2
11.9
± 0
.21
7.2
± 0
.5
7.8
± 0
.26
8.4
± 0
.20
C18:3
38.8
± 1
.36
39.6
± 0
.99
36.5
± 0
.66
35 ±
0.8
5
SF
A22.1
± 0
.80
31.3
± 1
.36
32.1
± 1
.88
31 ±
1.2
5
UF
A77.9
± 0
.85
68.7
± 1
.42
67.9
± 1
.88
69 ±
1.2
5
Bio
mas
s co
nte
nt(
dry
wei
gh
t, g
/L)
0.4
90
.74
11
.03
Ex
trac
ted
org
anic
co
nte
nt
(%)
35
.73
7.1
32
.33
0.3
TF
AM
E c
on
ten
t (%
) 1
1.2
6 ±
0.7
4
10
.23
± 0
.37
1
0.7
1 ±
0.5
9
8.8
1 ±
0.0
8
AcceptedPrepri n
tT
able
4
Fat
ty a
cid
co
mp
osi
tio
n (
%)
12
hr
15
hr
24
hr
C16:0
23.4
± 0
.61
24.8
± 0
.38
27.3
± 0
.25
C16:1
1.8
± 0
.06
1.6
± 0
.06
1.1
± 0
.06
C16:2
2.7
± 0
.06
1.9
± 0
.06
0.8
± 0
.06
C16:3
9.3
± 0
.12
9.4
± 0
.10
7.4
± 0
.12
C18:0
0.6
± 0
.06
0.7
± 0
.06
1.7
± 0
.06
C18:1
14.9
± 0
.4
15.7
± 0
.15
21.4
± 0
.21
C18:2
16.5
± 0
.12
16.8
± 0
.10
15.6
± 0
.10
C18:3
30.9
± 0
.46
29.1
± 0
.21
24.7
± 0
.1
SF
A24 ±
0.6
4
25.5
± 0
.36
29 ±
0.2
6
UF
A76 ±
0.7
2
74.5
± 0
.36
71 ±
0.2
6
Bio
mas
s co
nte
nt
(dry
wei
gh
t, g
/L)
1.1
1.3
11
.59
Bio
mas
s p
rod
uct
ivit
y (
g/L
/day
)
0
.07
90
.09
40
.11
4
Ex
trac
ted
org
anic
co
nte
nt
(%)
37
.83
8.5
39
.6
TF
AM
E/D
W (
%)
8.3
2 ±
0.6
8
10.4
4 ±
1.2
3
10.2
6 ±
0.6
5
24
AcceptedPrepri n
t T
able
5
Fat
ty a
cid
co
mp
osi
tio
n
Bef
ore
N-d
epri
vat
ion
A
fter
N-d
epri
vat
ion
C16:0
25.9
7 ±
0.8
5
28.2
± 0
.1
C16:1
2.2
± 0
.26
2.0
3 ±
0.1
2
C16:2
12.4
7 ±
0.3
1
4 ±
0.1
C16:3
02.5
7 ±
0.0
6
C18:0
00
C18:1
21.0
3 ±
0.5
9
32.6
3 ±
0.0
6
C18:2
11 ±
1.1
41
11.9
7 ±
0.1
5
C18:3
27.3
3 ±
1.1
5
18.6
3 ±
0.1
SF
A25.9
7 ±
0.8
5
28.2
± 0
.1
UF
A74.0
3 ±
0.8
5
71.8
± 0
.1
Bio
mas
s co
nte
nt
( d
ry w
eig
ht,
g/L
) 0
.56
1.2
4
Ex
trac
ted
org
anic
co
nte
nt
(%)
35
.35
0
TF
AM
E/D
W(%
)6
.22
± 0
.55
1
8.0
5 ±
0.9
0
25
AcceptedPrepri n
tTable 6
% Normal Starvation
FFA/DW 9.38% 2.16%
TG/DW 0.88% 9.47%
DG/DW 0.50% 1.08%
MG/DW 1.56% 7.67%
Total/DW 12.31% 20.38%
AcceptedPrepri n
t
Figure 1
27
AcceptedPrepri n
t
Figure 2
28
AcceptedPrepri n
t
Figure 3
29
AcceptedPrepri n
t
Figure 4
30